Peptides having xenopsin-like pharmacological activity

ABSTRACT

New penta- and octapeptides are provided represented by the formula: R-Arg-Pro-Trp-Ileu-Leu-OH wherein R stands for hydrogen atom or the grouping of pyrGluLys-Ileu-Leu-OH. Said peptides possess various pharmacological activities such as contracting activity on muscle of stomach fundus, depression of blood pressure and the like, and may be used in therapeutic treatments as substitute of the known octapeptide hereinafter-named &#39;&#39;&#39;&#39;Xenopsin&#39;&#39;&#39;&#39; obtained from natural source.

United States Patent Uchiyama et a1.

PEPTIDES HAVING XENOPSIN-LIKE PHARMACOLOGICAL ACTIVITY Inventors: Mikio Uchiyama, Urawa; Shinro Tachibana, Narashino; Kengo Araki, Niiza; Takafumi Nakamura, Tokyo, all of Japan Assignee: Eisai Co., Ltd., Tokyo, Japan Filed: Oct. 22, 1974 Appl. No.: 517,107

Foreign Application Priority Data Oct. 23, 1973 Japan 48-118550 Oct. 23, 1973 Japan 48-118551 US. Cl. 260/112.5 R; 424/177 Int. Cl. C07C 103/52; A61K 37/00 Field of Search 260/112.5

References Cited OTI-[ER PUBLICATIONS Erspamer, Ann. Rev. Pharm, 11, 337-347 (1971).

[ Dec. 23, 1975 Sander et al., Ann. Rev. Pharm., 12, 250-255 (1972).

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J. Suyat Attorney, Agent, or Firm-Werideroth, Lind & Ponack [57] ABSTRACT New pentaand octapeptides are provided represented by the formula:

R-Arg-Pro-Trp-lleu-Leu-OH 3 Claims, No Drawings PEPTIDES HAVING XENOPSIN-LIKE PHARMACOLOGICAL ACTIVITY This invention relates to new pentapeptide and octapeptide represented by the general formula:

R-ArgPro-Trplleu-Leu-OH wherein R is H or the grouping, pyrGluLys-Gly.

It has been found that the peptides of the formula (I) possess various pharmacological activities especially activities of contraction on muscle of stomach fundus (fundus ventriculi muscle), blood pressure depression and the like.

The present inventors formerly succeeded in isolation of a new peptide compound by extraction from skin of African frog (Xenopus laevis pipidae) and con firmed that said peptide compound possesses pharmacological activities such as contraction on muscle of stomach fundus, depression of blood pressure and the like, and a patent application (Japanese Patent application No. 82,103/72), was filed therefor.

On successive study of chemical structure, it has been confirmed that said new peptide is an octapeptide consisting of a series of the amino acids represented by and the compound was named Xenopsin.

In further study of the present inventors with respect to the pharmacological activities represented by said octapeptide, it has been confirmed that the pharmacological activities of Xenopsin rely upon the presence of the pentapeptide moiety represented by the formula:

ArgPro--Trplleu-Leu-OH which constitutes the essential part of the molecule of said octapeptide. It is therefore recognizable that the peptide compounds of the present invention, which are shown by the aforementioned formula (I), that is:

R-Arg-Pro-Trp-lleuLeu-OH (I) wherein R is H or the grouping of pyrGlu-LysGly, because of their chemical structures, should naturally have the Xenopsin-like phannacological activities. Actually, it has been confirmed that said compound of the formula (I) wherein R is hydrogen atom, and is thus the pentapeptide represented by the formula:

HArg-Pro--Trp-IleuLeu-OH (III) which belongs to one of the peptides claimed in the present invention possesses the pharmacological activities almost equivalent to 1/200 of those presented by the natural octapeptide of the formula (II), that is, Xenopsin.

In obtaining Xenopsin, there is, however, a decisive difficulty in that not only there is a limitation in com mercial viewpoint to avail African frog as natural source, but also it requires the complicated extraction steps for isolation of the intended compound from said natural source. In synthetic method of said compound, on the other hand, there is a defect of complication in the steps of successive conjunctions of the eight(8) amino acid units different from one another.

Contrary to the abovementioned synthetic method, the synthesis of the pentapeptide compound of the formula (III) according to the present invention can be effected more easily than the synthesis of Xenopsin. Since said pentapeptide is obtained by the steps of successive'conjunctions of only five() different amino acid units, it may be said that said excellent pharmacological activities represented by Xenopsin, that is, the minimum quantity required for representation of effective pharmacological activity is only lO*"lO' g/ml will be compensated by substitution with the new compound of the present invention, although the latter compound possesses lower pharmacological activities, if the simplicity of the total steps required for synthesizing the new pentapeptide compound is taken into consideretion.

In case where R is the abovementioned formula (I) is the grouping of pyrGluLys -Glu, said compound is an octapeptide having the formula:

which, as is evident from the formula, is the compound different from the octapeptide compound of the abovementioned formula (II), i.e., Xenopsin, in that the conjunctions of the second and third aminoacid groupings are in reverse order. It has also been found that the octapeptide compound of the formula (IV) shows the pharmacological activities such as contraction effect on muscle of stomach fundus, depression of blood pressure and the like equivalent to those of Xenopsin. The new octapeptide of the invention may thus be employed in leu of Xenopsin.

The pentapeptide of the formula (III) of the present invention may be prepared by splitting off by means of chemical treatment of the protective radical or radicals contained in the protected pentapeptide of the formula:

R,( N R )ArgPro---Trp--IleuLeu-OR=, (V) wherein R R and R are hydrogen atoms or one or more protective groupings capable of being split off by means of chmical treatment with the proviso that there is no case where all of R R and R are simultaneously hydrogen atoms.

Octapeptide compound of the abovementioned formula (IV), which belongs to the claimed peptides of the present invention, on the other hand, may be prepared by splitting off of the protective radical or radi- In the abovementioned formula (VI), R R R and R are hydrogen atoms or one or more of them are the protective radical(s) which is/are capable of being split off by chemical treatment that does not affect the octapeptide chain, i.e.,

of said octapeptide, with the proviso that there is no case where all of the radicals R R R and R are simultaneously the hydrogen atoms.

There are several types of said amino protective radicals R R and R in the formula (V); and R R R, and R in the octapeptide of the formula (VI), such as acyl-type radicals includding formyl, benzoyl, phtharyl, trifluoroacetyl and tosyl radicals; alkyl-type radicals including trityl, benzyl and the like; alkyliden radicals; and urethane-type radicals such as benzyloxycarbonyl, p-bromo-, p-chloro* and p-methoxy-benzyloxycarbonyl, tolyloxycyclohexyoxyand t-butoxycarbonyl radicals. As for the protective radical on the carbonyl, that is, R in the formula (V); or R in the formula (VI), there may be mentioned lower alkyl, such as methyl, ethyl, propyl and the like; benzyl, p-bromo-, p-chloro-, p-methoxy-benzyls and the like.

The splitting off of abovementioned protective radical or radicals from the protected pentaand octapeptides of the formulae (V) and (VI) by the chemical treatment according to the present invention can be effected by means of the customary methods such as treatment with trifluoroacet-ic acid,hydrofluoric acid and the like, as well as a catalytic reduction in the presence of palladium-carbon or the like.

In an embodiment, benzyloxy carbonyl and nitro groups, for example, may be 'split off by catalytic reduction in the presence of palladium carried on carbon as the catalyst in a solvent such as methanol, ethanol, dioxan, dimethyl formamide or the like; or by utilization of the hydrogen fluoride method disclosed in Bulletin of the Japanese Chemical Society 40, 2164 (1967); while t-butoxy-carbonyl as the protective radical may be split off by the treatment with trifluoroacetic acid. The resulting compound of the formula (I), if desired, may be converted into its pharmacologically acceptable acid-addition salts in accordance with a conventional salt-forming method. As pharmacologically acceptable inorganic acid, there may be mentioned hydrochloric, sulfuric, hydrobromic and nitric acids, for example; and as for the pharmacologically acceptable organic acid, there may be mentioned for mic, oxalic, acetic, citric, tartaric, fumaric, maleic and methane-sulfonic acids and the like.

The protected pentapeptide and octapeptide of the formulae (V) and (VI), which serve as the starting materials for carrying out the process of the present invention are new substances. Several methods may be utilized for the syntheses of the new protected peptides of the formulae (V) and (VI), depending upon the characteristics of the structural formulae of the purposed peptides. In one exemplification, said peptide may be obtained by condensation of the following two fragmental components A and B:

HTrplleul.cuOR (Fragment A).

(Fragment B),

wherein R,, R and R have the meanings same as those aforementioned.

These fragments A and B may together be condensed to form the compound of the formula (V) according to the methods usually employed for peptide-conjunction, such as Merifield method, azide method, mixed acid anhydrides method, carbodiimide method and activated ester method, and W. Konig and R. Geigers method (a modification of the aforementioned carbodiimide method in the presence of N-hydroxyimide compound).

Solvent for the condensation reaction may suitably. be employed such as amide series compounds including dimethyl formamide, dimethyl acetamide and the like; halogenated alkyl series solvent such as dichloromethane, chloroform, and carbon tetrachloride; ether series solvents such as ethyl ether; tetrahydrofuran and dioxane, alone or in an admixture thereof.

The fragment A is also a new compound which may be prepared by successive bondings of the respective amino acid units. For example, the fragment of the formula:

HTrplleul.cu-OBZL (B') may be prepared in accordance with the following FIG.

BOC OBZL TFA F igure 1 Continued have the following meanings:

and

(Fragment D), wherein R R R and R have the meanings same as those aforementioned.

The condensation of the Fragments C and D to form the compound of the formula (VI), like the synthesis of the protected pentapeptide as aforementioned, may be effected in accordance with the known methods usually employed for the peptide conjunction such as Merifield method, azide method, mixed acid anhydride method,

carbodiimide method, activated ester method and W.

Konig and R. Geigers method (a modification of the abovementioned carbodiimide method in the presence of N-hydroxyimide compound).

Solvent for the condensation reaction may suitably be employed such as amide series solvents including dimethyl formamide, dimethyl acetamide and so on; halogenated alkyl series solvents such as dichloromethane, chloroform and carbon tetrachloride;.ether series solvents such as ethylether; tetrahydrofuran and dioxane, alone or in an admixture thereof.

The abovementioned fragments C and D are also new .compounds and may be prepared by successive bondings of the respective amino acid units. For example,

one of the Fragment A having the following formula: 7.pyrGlu(N Z)LysGly-(N -NO2. )Arg-ProOH (C) may be prepared in accordance with the following FIG. ll.

LII

Figure ll pyrGlu Lys Gly Arg P 0 N0 BOC H OBZL g OH l BOC OBZL v N0 BOC OH H' OBZL l N0 BOC 0821.

Z L No In the above formula B and FIG. I, the abbreviations I Figure ll-continued BOC H 0871.

(2) z HOBT o BOC OBZL z Z- ONP H OBZL z o 7. 3: 2 OBZL z 0 z /N CH Further, one of the fragment D, the protected tripeptide having the following formula, for example,

HTrpIlel.euOBZl. (0') may be prepared in accordance with the following FIG. III:

Figure Ill Trp lleu L eu BOC-OH H---OBZL (2) HOW. DCC BOC OBZI.

TFA BOC- ()H H OBZL (4) BOC OBZL V TFA I-' 01321.

In the abovementioned fonnulae (C) and (D), and FIGS. II and III, the abbreviations have the following meanings:

( l Mixed acid anhydrides method;

(2): W. Konig and R. Geigers method;

(3): Activated ester method; and

(4): Carbodiimide method;

Z: Benzyloxycarbonyl ONP: p-Nitrophenyl ester BOC: t-Butoxycarbonyl OBZL: Benzyl ester TFA: Trifluoro acetic acid HOBT: N-Hydroxybenzotriazole DCO: Dicyclohexylcarbodiimide It has been found that the pentapeptide of the formula (III) obtained in accordance with the present invention, as the result of animal tests, possesses the pharmacological activities equivalent to 1/200 of those of Xenopsin.

The octapeptide compounds of the formula (IV) obtained by the process of the present invention, on the other hand, show in animal test the following pharmacological effects:

1. Test for contraction on muscle of stomach fundus of rat (Magnus method):

Threshold dose: 0.1 ng/ml Dose response: observed p 2. Test on rat for blood pressure depressing effect:

Threshold dose (intravenous injection): 1.2 g/ml.

From the data, it is apparent that the peptide compounds of the present invention are useful as blood pressure depressant or the like in therapeutic treatments.

a 6 The following examples will serve to illustrate the invention.

EXAMPLE 1 Synthesis of HsArg-Pro-TrpIleu--LeuOH 1.1 Grams of BOC-(N -NO )ArgProTrpIleuLeuOBZL are dissolved in 50 ml of 98% formic acid. The resulting solution is stirred at room temperature for 4 hours, which is then concentrated under reduced pressure.

To the concentrate, there is added 50 ml of ethylether and precipitate separates out is recovered by filtration.

The precipitate is dissolved in ml of a 10% acetic acid in methanol, which is then aubjected to catalytic reduction in the presence of 10% palladium on carbon for 2 days. After completion of the reduction, the spent catalyst is removed by filtration. The filtrate is concentrated under reduced pressure. To the concentrate are added ethylether and the precipitate separates out is recovered by filtration and washed with ethyl ether.

There are obtained 08 grams of the purposed crude product. The crude product is purified chromatographically with a column charged with SP-Sephadex (a commercial product sold by Pharmacia Company) passing through 0.05 M ammonium formate buffer solution at pH 6.5 as eluting liquor. The main flow-out liquor fraction recovered is subjected to freeze-drying to obtain the purposed product in a form of formate having the following characteristics.

Analytical data of aminoacid units:

Arg=0.96, Pro=l0l, Ileu=l.00, Leu=0.97

Elementary analysis for C l-I N O .2HCOOH.2H O

gives:

C H N Calculated (Va)! 53.24 7.5:: 15.52 Found a); 53.15 7.23 l5.83

Thin layer silica gel chromatography the starting material employed, is obtained as follows:

575 mg of l-I-Trp-lleu-Leu-OBZL hydrochloride are dissolved in 10 ml of tetrahydrofuran. To the resulting solution, there are added 1.1 ml of a 10% N-methylmorpholine tetrahydrofuran solution, followed under stirring with cooling at 0C. by the successive additions of 416 mg of BOC-(N' NO )Arg- ProOl-l, 250 mg of N-hydroxybenzotiazol and 250 mg of dicyclohexylcarbodiimide. The whole is stirred at the temperature same as the above for 5 hours and settled for overnight at room temperature. The precipitate separated out is removed by filtration, and the filtrateis concentrated under reduced pressure. The resulting concentrate is dissolved in 100 ml of ethyl acetate, and the solution is successively washed with an aqueous normal solution of sodium bicarbonate, water, M/4 citric acid, water, again anaqueous normal solution of sodium bicarbonate and water, dried on anhydrous sodium sulfate.

C H N Calculated (71); 59.52 7.29 15.09 Found ("/z )1 59.67 7.26 14.78

In thin layer silicalgel chromatography:

Rf=0.36 (in developing medium ofcthylacetate). and

Rf=().92 (in developing medium of n-hutanol acetic acid water=4z l :5)

H-Trp-lleLeu-OBZL used as starting material in Example 1 is also a new compound which may be obtained as follows:

REFERENTIAL EXAMPLE 1 Synthesis of H-Trp,Ileu LeuOBZL a. BOCTrpl leu--LeuOBZL, synthesis of; 3.9 g of BOCll'euLeuOBZL are dissolved in a 50% trifluoroacetic acid/dichlorometliane solution. The resulting solution is allowed to stand for 1.5 hours at room temperature, concentrated under reduced pressure; and the residue is added to 200 ml of hydrogenchloride-containing ethylether. White precipitate separated out is recovered by filtration and dried. 3 Grams of the dry precipitate having the melting point of 154-155C. are dissolved in 30 ml of tetrahydrofuran, and 0.88 mlof N-methylmorpholineare further added thereto. Under cooling to 0C., there are added successively 2.4 grams of BOCTrpOH, 1.3 grams of N- hydroxybenzotriazol and 1 .8 grams of cyclohexylcarbodiimide. After stirring at that temperature, for 1 hour, furtherstirring is continued, for 4 hours and allowed to stand for overnight. The precipitate crystallized out is removed by filtration, and the filtrate, after washing with etl ylacetate, is concentrated .under reduced pressure. The solution obtained by dissolving the residue in ethyl ester is washed successively with water, an aqueous normal solution of sodium bicarbonate, again water, M/4 citric acid aqueous solution and still again water; and dried on Glaubers salt. The residue obtained by concentration under reduced pressure is recrystallized from a mixture of ethyl ether and n-hexane. There are thus obtained 4.5 grams of the purposed product in a white powdery sdbstance having the melting point of l54l56C.

Value of mass-spectrum: n/e 620 (M Elementary Analysis for C i-1 N 0 gives:

1 c H N Calculated (/r): 67.70 7.81 9.03 Found 67.73 8.06

In thin layer silica gel chromatography: Rf=0.86 (in ethyl ether as developer) b. REFERENTIAL EXAMPLE (b) Synthesis of H-Trplleu-Leu-OBZL 2.5 Grams of BOCTrp--lleuLeu-OBZL are dissolved in ml of a 50% trifluroacetic acid/dichloromethane solution containing 0.4 ml of mercaptoethanol and 0.5 ml of anisol. The resulting solution is stirred at room temperature for 30 minutes, and it is concentrated under reduced pressure to 5 ml. To the concentrate are successively added 100 ml of ethyl ether and 4 ml of a 10%-hydrogen chloride/ethylacetate solution.

A gelatinous precipitate is recovered by filtration to obtain the purposed product in a form of hydrogen chloride addition salt which is recrystallized from a mixture of ethanol/isopropyl. ether. Yield 1.9 grams, arid the melting point of 2l9220C.

[a],, =25.33 (c=0.75 in 50% methanol) Elementary analysis for C H N O HCLH O gives:

1 30 6: 20 p24 as developer) EXAMPLE 2 Synthesis of Pyr GluLys-GlyArgPro-Trp-lleuLeu-OH 140. mg of A-Pyr Glu-(N Z)LysGly- (N"NO )ArgPro -eTrp-IleuOBZL are dissolved in 2'5 ml of methanol which contains 0.05 ml of acetic acid. The solution is subjected to catalytic reduction in the presence of a 10% palladium carried on carbon for 9 days.continuously. After the treatment, the spent catalyst is filtered off. The filtrate is concentrated under reduced pressure. The resulting residue 18 purified on a column packed with SP- Sephadex (a commercial product sold by Pharmacia Company) passing through ammonium formate buffer solution at pH 6.5 as eluting liquor. The collected main flow-out liquid fractions is subjected to freeze-drying toobtam thepu'rposed product in a white powder.

[a],, =4l.7 (c=0.43 in a mixture of methanol and dimethyl formamide in a ratio of l 0.1)

Elementary analysis for C H N O gives:

e i H N Calculated 53.43 6.06 15.58 Found (72): 53.57 5.90 15.40

Thin layer silica gel chromatography gives; Rl'-0.47 (in developing medium consisting of nbutanol acetic acid water 4 l 5), and Rf=0.76 (in developing medium consisting of nbutanol acetic acid :pyridine water =30 2O 6 24). l p v The starting material, Z-PyrGlu(N Z)Lys-Gly- (N" NO2. v

' ;Arg-ProTrp---lleu---LeuOBZE 1 employed in the above experiment is obtained as follows:

690 mg of l-l-Trplleu-LeuOBZL.HCl are dissolved in 20 ml of dimethyl formamide. To the solution, there are added 0.168 ml of triethylamine and then 1.0

gof

and the whole is stirred under cooling to 10C. To the solution, 200 mg of hydroxybenzotriazol and 258 mg of dicyclohexylcarbodiimide are added, and the reaction is conducted at the temperature of 10C. for 5 hours, followed by stirring at room temperature for overnight. The reaction mixture is further allowed to stand at room temperature for one day. Precipitate crystallized out is removed by filtration and the filtrate is concentrated under reduced pressure. 80 ml of ethyl ether are added to the concentrate and oily substance is recovered by decantation, which is then dissolved in 3 ml of tetrahydrofuran. 100 ml of ethyl acetate is added to the solution, and the precipitate separated out is recovered by filtration. 1.1 Grams of the crude product are thus obtained. The crude product is recrystallized twice from a mixture of dimethyl formamide/ethylacetate to obtain the intended product in a pure form having the following characteristics:

[a],, =37 (c=1 in methanol) Elementary analysis for C I-1 N gives:

Calculated (%)I 52.22 6.73 17.13 Found ("/z): 52.11 6.86 16.25

dures.

REFERENCE 1 Preparation of BOC(N NO )Arg- Pro-OBZL 4.7 Grams of BOC(N NO )Arg-Pro-OH are dissolved in 30 ml of dimethylformamide. To the resulting solution under cooling at 50C., there are further added drop by drop under stirring 1.65 ml of N-methylmorpholine and 2.1 grs. of isobutyl chloroformate. Continuing the stirring the solution further minutes, 4.35 grs. of H-ProOBZL.HCl, 1.98 ml of Nmethylmorpholine, both dissolved in ml of dichloromethane are added dropwise under stirring. After stirring further 1.5 hours at 50C. the reaction mixture is allowed to stand for 12 hours at room temperature, and concentrated under reduced pressure. Resulting residue is dissolved in 150 m1 of ethylacetate. The solution iswashed successively with water, M/4 citric acid in water, again water, aqueous normal solution of sodium bicarbonate and still again water; dried on magnesium sulfate, and concentrated under reduced pressure. The residue is subjected to chromatography through column packed with Wako-Gel G100 (a commercial product of Wako Pure Chemicals Company) using ethylacetate as eluating solvent. The collected eluates are subjected to thin layer silica gel chromatography with ethyl acetate as developer. Portion of a 10 single spot at at Rf=0.4l is collected and recovered the purposed product from a tetrahydrofuran/ethyl ether system. Yield of the product is 5.0 grams. The product melts at about 50C. with froth decomposition.

[a] "*-6l (c=l in methanol) chloromethane solution, stirred at room temperature for 40 minutes and concentrated under reduced pressure to about 5 ml.

To the concentrate there are added 50 ml of ethyl ether, and the precipitate separates out is recovered by filtration, washed with ethyl ether and dried. 3.2 Grams of the dry substance are thus obtained.

Apart from the above, 1.08 grs. of BOCGlyOl-l are dissolved in 10 ml of tetrahydrofuran. To the resulting solution are added under cooling 0.68 ml of N- methylmorpholin and 0.84 grs. of isobutylchloroformate at 5-0C. After 15 minutes, there is added dropwise a solution prepared by dissolving the dry substance prepared in the preceding paragraph in a mixture of 0.68 ml of N-methylmorpholin and 15 ml of dichloromethane. The whole is stirred at the temperature same as that abovementioned for 2 hours and at room temperature overnight. After concentration under reduced pressure, the reaction product is dissolved in 200 ml of ethylacetate, washed successively with M/4 citric acid, water, n-aqueous solution of sodium bicarbonate and again water, dried on Glaubers salt and concentrated under reduced pressure to obtain oily substance. The substance is subjected to column chromatography through Wako gel C-l00 as carrier and ethyl acetate as eluating liquor and then thin layer silica gel chromatography in ethyl acetate as developing liquor to collect 3.0 grs. of the single spot portion at Rf=0.l4 and from which the purposed substance is recovered as an oily product.

c. Preparation of BOC-(NZ)LysGly 3 Grs. of BOCGly-(N NO )Arg- Pro-OBZL are dissolved in a mixture of 20 ml of a 50% trifluoro-acetic acid/dichloromethane and 1 ml of anisol, stirred at room temperature for 40 minutes and the resulting solution is concentrated under reduced pressure. To the residue thus obtained, there is added ml of ethyl ether containing 3 ml of a 10% hydrogen chloride/isopropyl ether. Precipitate separated out is recovered by filtration, washed with ethyl ether, dried to obtain 2.4 grs. of a dry substance consisting of HGly(N --NO )ArgProOBZL in a form of hydrogen chloride addition salt.

2.35 Grs. of said addition salt and 2.7 grs. of BOC- (N Z)Lys-OH.dicyclohexylamine salt are suspended in 50 ml of tetrahydrofuran and well shaken. Under cooling, there are added 0.94 g of Nhydrox- 1 1 ybenzotriazol and 1.67 g of dicyclohexyl urea. The whole is stirred at that temperature for three hours and then at room temperature overnight.

Precipitate separated out is removed by filtration and the filtrate is concentrated under reduced pressure. The residue is dissolved in 150 ml of ethyl acetate and the solution is washed successively with water, n-aqueous solution of sodium bicarbonate, again water, M/4 citric acid, still again water and n-aqueous solution of sodium bicarbonate and further water; dried on Glaubers salt, and concentrated under reduced pressure. The residue is reprecipitated from a mixture of ethyl acetate and ethyl ether. 2.7 Grs. of the precipitate are recovered which are recrystallized from a mixed solvent consisting of ethyl ether and peterolium ether. M. P. of the crystal is 5763C. with froth decomposition.

Elementary analysis of the product for C ,,H N O Thin layer silica gel chromatography gives: Rf=0.89 (in n-butanol acetic acid water in the ratio of4 1 5 as developer);-and Rf=0.31 (in ethylacetate ethanol in the ratio of 9 l as developer). (1. Preparation of BOC(NZ)LysGly- (N*NO )ArgProOH 2.5 Grs. of BOC(NZ)Lys-Gly(N"- NO )ArgPro-OBZL are dissolved in a mixture of 9 ml of methanol and 3 ml of n-aqueous solution of caustic soda, and the solution is stirred at room temperature for 5 hours. To the reaction liquor, there are added 2.5 ml of n-hydrochloric acid and the solution is concentrated under reduced pressure. The residue is extracted with ethylacetate, and the extract, after washed with water and dried on Glaubers salt, is concentrated under reduced pressure. To the residue, there is added ethyl ether and the precipitate separated out is recovered by filtration, which is then dissolved in ml of n-aqueous solution of sodium bicarbonate, washed with ethylacetate, acidified with M/4 citric acid and extracted with ethyl acetate. The extract is washed with water, dried on Glaubers salt and concentrated under reduced water. To the residue, there is added ethyl ether and the purposed product is recovered therefrom in a solid form. Yield 1.2 g; melting point is 92-95C (under froth decomposition) [a],, =37 (t=l in methanol) Elementary analysis for C H N O gives:

C H N Calculated 71. 52.22 6.73 17.13 Found (71): 52.11 6.86 16.25

1 Gr. of BOC(N Z)LysGly(N NO2) ArgPro-OH is dissolved in a mixture consisting of 0.2 ml of anisol and 15 ml of a 50% trifluoro-acetic acid/dichloromethane, and the solution, after stirred at room temperature for one hour, is concentrated under pressure. To the concentrate, there is added 45 ml of ethyl ether and the precipitate separated out is recovered by filtration, washed with ethyl ether and dried to obtain. 1 gr. of the dry substance, that is, trifluoroacetate of H(l I Z)LysGly(N' NO )Arg- ProOl-l.

The dry substance is dissolved in 9 ml of dimethyl formamide, and to the solution there is added 0.92 ml of N-methylmorpholine and 0.58 g of ZPyr- Glu-ONP. The precipitate separated out is recovered by filtration. The precipitate is reprecipitated from a mixed solvent of ethanol .and ethyl acetate, which is then reprecipitated twice from a mixed solvent of ethanol and water. There are obtained 0.9 g. of the powdery substance. Melting point of the product is l30150C. with decomposition.

[a],,"=41.7 (c=1 in methanol dimethylformamide in the ratio of l 0.1) Elementary analysis for C H N O H O gives:

a 50% trifluoro-acetic acid/dichloromethane solution, and the resulting solution, after allowed to stand at room temperature for 1.5 hours, is concentrated under reduced pressure. The residue is introduced into 200 m1 of ethyl ether which contains hydrogen chloride. White precipitate separated out is recovered by filtration and dried. The precipitate having the melting point of 154155C., which amounts to 3.0 grs., are dissolved in 30 ml of tetrahydrofuran, and 0.88 ml of N-methylmorpholine are then added thereinto. To the mixture, there are added 2.4 grs. of BOCTrp-OH under cooling to 0C. and then 1.3 grs. of N-hydroxybenzotriazol and 1.8 grs. of dicyclohexylcarboimide. The mixture after stirring at that temperature for one hour, additional stirring at room temperature is continued for 4 hours and then allowed to stand overnight. Precipitate separated out is removed by filtration and the filtrate after washing with ethylacetate is concentrated under reduced pressure. The residue is dissolved in ethylacetate, washed successively with water, naqueous solution of sodium bicarbonate, again water, M/4 citric acid in water and still again water and dried on Glaubers saft. The residue obtained by concentration under reduced pressure is recrystallized from a mixture of ethyl ether and n-hexane. There is thus obtained the contemplated product in a form of white powder. Yield: 4.5 grs., melting point: l54-156C.

[a],, =-4.2 (0 1 in methanol) and l3 14 mass spectrum: m/e 620 (M [a],, =25.33 (t=().75 in 50% ethanol) Elementary analysis for C; -,H N O, gives: Elementary analysis of the product,

C H N O HCLH O gives:

C H N v 5 c H N Calculated 7a); 67.70 7.81 9.03 Found (71 )1 773 8.06 9.34 Calculated 7, 62.63 7.55 9774 Found (/1): 62152 726 9.84

Thin layer silica gel chromatography gives: 10 (in ethyl ether as developer) Thin layer silica gel chromatography gives:

Rf=0.83 (in the developing solvent of n-butanol acetic acid water 4 l 5); and

Rf=0.87 (in the developing solvent of n-butanol acetic acid pyridine water 30 z 6 2O 34) What is claimed is:

b. Preparation of HTrpIleuLeu-OBZL 2.5 Grs. of BOCTrplleuLeu-OBZL are dissolved in 20 ml of a 50% trifluoroacetic acid/dichloromethane mixture, which contains 0.4 ml of mercapto ethanol and 0.5 ml of anisol. The solution, after 1 Fe tide Com Ounds re resented b the eneral stirring at room temperature for 30 minutes, is concenp p y g trated under reduced pressure to 5 ml. To the concentrate, there are first added 100 ml of ethyl ether and wherein R Stands f H or h grouping f pyrthen 4 ml of a 10%-hydrogen chloride/ethyl acetate G] L G] solution. Gelatinous precipitate thus separated out, 2, P tid ound claimed in claim 1 which is the hydrochloride of the purposed product, is wherein R is H. recovered by filtration and recrystallized from a mix- 3. Octapeptide compound claimed in claim 1 ture of ethanol/isopropyl ether. Yield: l.9 grs.; melting wherein R is the grouping of pyrGluLys-Gly. point: 219-220C. 

1. PEPTIDE COMPOUNDS REPRESENTED BY THE GENERAL FORMULA: R-ARG-PRO-TRP-LLEU-OH WHEREIN R STANDS FOR H OR THE GROUPING OF PYRGLU-LYS-GLY.
 2. Pentapeptide compound claimed in claim 1 wherein R is H.
 3. Octapeptide compound claimed in claim 1 wherein R is the grouping of pyrGlu-Lys-Gly. 